Abstract
<title>Abstract</title> <p> Background Arbuscular mycorrhizal (AM) fungi are obligate plant symbionts that form mutualistic associations with most terrestrial species. Previous studies centered on the model species <italic>Rhizophagus irregularis</italic> have reported that AM fungi possess a small number of ribosomal DNA (rDNA) copies with substantial intragenomic variation. We analyzed the genome and rDNA sequences of <italic>R. clarus</italic> HR1, a species closely related to <italic>R. irregularis</italic> to investigate the conservation and evolution of rDNA in AM fungi. Results Using long-read sequencing, we generated a highly continuous genome assembly of <italic>R. clarus</italic> HR1. Comparison of its genome structure and rDNA sequences with the chromosome-level genomes of <italic>R. irregularis</italic> showed that the overall genome structure was conserved between the two species, and that a non-tandem distribution of low-copy 45S rDNAs was also observed in <italic>R. clarus</italic> . Despite the absence of tandem repeat structures, most rDNA regions, especially 18S rDNA, are more conserved within species than between species. In contrast, conserved polymorphisms were identified in the D4 region of 26S rDNA and were shared between <italic>R. clarus</italic> and <italic>R. irregularis</italic> . These polymorphisms are predicted to be reflected in the structural variations of ES27L in 26S ribosomal RNA (rRNA) and may suggest potential functional variations in rRNA molecules. Phylogenetic and sequence-similarity analyses indicated that conservation patterns differed among regions within 45S rDNA, particularly between intraspecific and interspecific comparisons. Conclusions Our results demonstrate that AM fungi maintain rDNA sequences that are generally more conserved within species than between species despite possessing dispersed non-tandem rDNA copies. Simultaneously, some rDNA regions retained conserved polymorphisms between species, suggesting heterogeneous patterns of rDNA evolution within the 45S rDNA unit. Our results suggest that concerted evolution does not occur uniformly across the entire rDNA region of AM fungi. In addition, the observed intragenomic diversity of rDNA highlights the potential limitations of species identification and taxonomic description based solely on multicopy rDNA marker sequences. Our results provide genomic information for <italic>R. clarus</italic> , a species suitable for comparative genomic studies with the model AM fungus <italic>R. irregularis</italic> , and contribute to a better understanding of AM fungal genome characteristics and the evolution of rDNAs. </p>